|
| Status |
Public on Oct 30, 2013 |
| Title |
WT in xylose, replica 1 (14308825) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
[test] S288c
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype/variation: WT
carbon source: xylose (SX)
|
| Growth protocol |
For S.cerevisiae microarrays, starter cultures of S288c strain were in raffinose medium (SR) as carbon source, and grown at 30C to an OD600 of 0.7 - 0.8. The cells were then washed with sterile water. The cell pellet was resuspended in S-based medium (no sugar) and an equal amount of cells transferred in no sugar (S) or xylose (SX) medium for experiments samples or in glucose (SD) medium for the reference samples. The cultures was incubated for ~2h until an increase in the OD600 was observed for the reference culture in glucose. For C.albicans microarrays, starter culture of SC5314 was in SD (glucose) medium. The overnight culture was washed with water and used to inoculate xylose (SX) or glucose (SD) medium at an OD600 of 0.1 and grown at 30C for ~5-6 h to an OD600 of ~0.8 .
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by mechanical disruption of the cells with glass beads using the FastPrep-24 (MP Biomedical). Thereafter, total RNA was isolated and purified with the Rneasy kit (Qiagen cat.74104).
|
| Label |
cy5
|
| Label protocol |
Indirect labeling with Cy3 and Cy5 was performed. For expression profiling experiment, 20 µg of total RNA was reverse transcribed using oligo(dT)21 in the presence of amino-allyl-dUTP (Sigma A-410) and Superscript III reverse transcriptase (Invitrogen). Thereafter, template RNA was degraded by adding 2.5 units of RNase H (USB cat.70054Z) and 1 ug of RNase A (Pharmacia) followed by incubation for 15 min at 37°C. The cDNAs were purified with QIAquick PCR Purification Kit (Qiagen) with 5mM potassium phosphate pH8.5 replacing the tris in the wash and elution buffers. The cDNAs were labeled with mono-reactive dyes (GE-Healthcare/ Amersham: cy3 cat. PA23001, cy5 cat. PA25001). The labeled DNA was purified with QIAquick PCR Purification Kit (Qiagen).
|
| |
|
| Channel 2 |
| Source name |
[reference] S288c
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype/variation: WT
carbon source: glucose (SD)
|
| Growth protocol |
For S.cerevisiae microarrays, starter cultures of S288c strain were in raffinose medium (SR) as carbon source, and grown at 30C to an OD600 of 0.7 - 0.8. The cells were then washed with sterile water. The cell pellet was resuspended in S-based medium (no sugar) and an equal amount of cells transferred in no sugar (S) or xylose (SX) medium for experiments samples or in glucose (SD) medium for the reference samples. The cultures was incubated for ~2h until an increase in the OD600 was observed for the reference culture in glucose. For C.albicans microarrays, starter culture of SC5314 was in SD (glucose) medium. The overnight culture was washed with water and used to inoculate xylose (SX) or glucose (SD) medium at an OD600 of 0.1 and grown at 30C for ~5-6 h to an OD600 of ~0.8 .
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by mechanical disruption of the cells with glass beads using the FastPrep-24 (MP Biomedical). Thereafter, total RNA was isolated and purified with the Rneasy kit (Qiagen cat.74104).
|
| Label |
cy3
|
| Label protocol |
Indirect labeling with Cy3 and Cy5 was performed. For expression profiling experiment, 20 µg of total RNA was reverse transcribed using oligo(dT)21 in the presence of amino-allyl-dUTP (Sigma A-410) and Superscript III reverse transcriptase (Invitrogen). Thereafter, template RNA was degraded by adding 2.5 units of RNase H (USB cat.70054Z) and 1 ug of RNase A (Pharmacia) followed by incubation for 15 min at 37°C. The cDNAs were purified with QIAquick PCR Purification Kit (Qiagen) with 5mM potassium phosphate pH8.5 replacing the tris in the wash and elution buffers. The cDNAs were labeled with mono-reactive dyes (GE-Healthcare/ Amersham: cy3 cat. PA23001, cy5 cat. PA25001). The labeled DNA was purified with QIAquick PCR Purification Kit (Qiagen).
|
| |
|
| |
| Hybridization protocol |
The microarray slides were pre-hybridized for 2 hours at 42°C with DIGeasy hybridization buffer (Roche) containing 0.5 ug/ul of yeast tRNA (Roche) and 0.5 ug/ul of salmon sperm DNA (Invitrogen) and subsequently washed with 0.1X SSC and air dried. The slides were then hybridized with labeled cDNAs overnight at 42°C in DIGeasy hybridization buffer with yeast tRNA and salmon sperm DNA. Hybridization was done in a "SlideBooster" chamber (Advalytik)
|
| Scan protocol |
ScanArray Gx scanner (Perkin Elmer) at 10-µm resolution
|
| Description |
Sample name: 14308825.txt Sc
|
| Data processing |
Expression array data were normalized as following : Signal intensity was quantified with QuantArray software (Perkin Elmer) and final Lowess normalization and inspection of the data was done with the GeneSpring package GX v.7.3 (Agilent Technologies).
|
| |
|
| Submission date |
Aug 30, 2013 |
| Last update date |
Oct 30, 2013 |
| Contact name |
Daniel Dignard |
| E-mail(s) |
daniel.dignard@cnrc-nrc.gc.ca
|
| Organization name |
NRC
|
| Department |
HHT
|
| Street address |
6100, Royalmount
|
| City |
Montreal |
| State/province |
QC |
| ZIP/Postal code |
H4P 2R2 |
| Country |
Canada |
| |
|
| Platform ID |
GPL13945 |
| Series (1) |
| GSE50476 |
Comparative xylose metabolism among the ascomycetes C. albicans, S. stipitis and S. cerevisiae |
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